(A) Study pathogenesis and pathophysiology of high biocontainment viral pathogens utilizing molecular technologies including reverse genetics systems: A major achievement of the past fiscal year is the establishment and characterization of the first nonhuman primate disease model for Crimean-Congo hemorrhagic fever virus (CCHF). Utilizing the Hoti strain, Cynomolgus macaques do develop moderate to severe disease that is fatal in approximately 20% of the animals upon a combination of subcutaneous and intravenous inoculation. Disease progression is very like human disease with most hallmark signs being displayed. The model will be extremely valuable for pathogenesis studies and countermeasure development. Further characterization such as a serial sacrifice study is ongoing. (manuscript submitted) We further studied the pathogenicity of multiple Ebola virus, strain Makona, isolates. The study was initiated after in vitro reports that Makona accumulated mutations in the glycoprotein gene that were associated with adaptation to humans during the West African Ebola epidemic. Due to our involvement in Liberia and Mali we have a unique set of Ebola-Makona isolates covering a larger portion of this outbreak. We tested different isolates displaying or lacking the questionable mutations utilizing a mouse and the rhesus macaque model. Surprisingly, all isolates behaved very similarly independent of the genotype, causing severe or lethal disease in mice and macaques, respectively. Likewise, we could not detect any evidence for differences in virus shedding. Thus, no specific biological phenotype could be associated with these EBOV-Makona mutations in vivo. (manuscript under review) We have continued to utilize humanized mice to study ebolavirus pathogenesis and immune responses. To investigate differences in viral pathogenicity, humanized mice (hu-NSG-SGM3) were inoculated with Ebola virus (EBOV, causes fatal human disease) or Reston virus (RESTV, does not cause disease in humans). Consistent with differences in disease in human infection, pronounced weight loss and markers of hepatic damage and disease were observed exclusively in EBOV-infected mice. These abnormalities were associated with significantly higher EBOV replication in the liver but not in the spleen, suggesting that efficiency of viral replication in select tissues early in infection may contribute to differences in viral pathogenicity. (manuscript under revision; studies ongoing) (B) Study immune responses to infection and vaccination of high containment viral pathogens and develop new vaccine candidates: The VSV vaccine efforts are reported under the 'Trivalent Filovirus Vaccine' project. In the meantime, we have proceeded with the development of alternative vaccine candidates for filoviruses. One approach is targeted towards wildlife vaccination, in particular the great apes, using the concept of a disseminating vaccine vector. The idea is the introduction of a recombinant CMV expressing the Ebola virus glycoprotein. Proof-of-concept studies have been successful in the mouse and rhesus macaque model using a murine and rhesus CMV vector, respectively. We have also continued to investigate a vaccine based on Modified Vaccinia Ankara (MVA) expressing filovirus-like particles; initial rodent and nonhuman primate studies have revealed very promising results. (manuscript in preparation; studies ongoing) (C) Study vector/reservoir transmission of high containment viral pathogens using appropriate animal models: We have studied infection kinetics of different Lassa virus in the Mastomys reservoir utilizing a unique colony established here at RML. The animals support virus replication and shedding for several weeks before Lassa virus gets cleared. The model will allow for important transmission studies. (manuscript in preparation; studies ongoing) (D) Utilize in vitro and in vivo systems to study the interactions between viral pathogen or viral components and host cells and develop new antiviral strategies: Previously we had tested the efficacy of two antivirals, T-705 and ribavirin, in the guinea pig model of Lassa virus infection. T-705 showed promise as a treatment for Lassa virus with protection even when treatment was started post-disease onset. This year, we could demonstrate efficacy of T-705 against Lassa infection in the nonhuman primate model. (manuscript in revision) (E) Study the epidemiology and ecology of high biocontainment pathogens utilizing newly developed rapid, sensitive and specific diagnostic test systems including those that can be applied under field conditions: From August 2014 through May 2015 we have operated a diagnostic laboratory in Monrovia, Liberia to support the international response to the West African Ebola epidemic. We have continued to analyze data and demonstrated the importance of performing on-line clinical chemistry and malaria differential diagnostics. Most importantly, we have identified the interesting observation that plasmodium co-infection increases the survival rate of Ebola-infected patients. We have started animal studies (mouse model) to better understand the underlying mechanism. (study ongoing)